siRNA/RNAi/dsRNA

At metabion, we consider RNA oligomer duplex production and delivery an "add-on" to the service-value chain based on our unmatched capability to produce high quality ssRNA oligos. In most cases dsRNA is used for siRNA allocations.

siRNA is well studied in the RNA interference (RNAi) pathway, where it regulates the expression of specific genes, whose mRNA bears complementary nucleotide sequences. In this context, siRNAs function by causing the disruption of complementary mRNAs, resulting in no translation. siRNAs also act in RNAi-related pathways, like in antiviral mechanisms or in shaping the chromatin structure of a genome. The complexity of these pathways is only now being elucidated. However, synthetic siRNAs have already become an important tool to gene validation or drug targeting as, at least in principle, any gene can be knocked down by a synthetic siRNA.

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-30 base pairs in length. siRNAs have a well-defined structure: short double-stranded RNA (dsRNA) with phosphorylated 5' and hydroxylated, two nts overhanging 3' ends.

Average in-house turnover 5-10 working days (freight forwarders delivery time not included) and refer to our standard portfolio. In terms of "counting" working days, orders placed past 3 pm German time are considered to be next day's order.

Additionally, we offer a seasoned selection of suitable backbone and/or base modifications, which can be introduced internally and/or at both ends (3´ and/or 5´). Among these are:

Each siRNA molecule is synthesized as two ssRNA oligonucleotides between 11 and 40 nucleotide in length, which are then aligned to form a duplex. The siRNA duplex typically has 2 DNA nucleotides overhanging at each 3’-end. For more information about RNA synthesis, read our RNA FAQs.

Each ssRNA strand is HPLC purified as well as mass-checked for best quality.

Synthesis scale refers to the amount of starting CPG (controlled-pore glass) support-bound monomer used to initiate the DNA synthesis, not the amount of final material synthesized. This is the same for all manufacturers of synthetic DNA using standard phosphoramidite chemistry. When a synthesis scale of 40 nmole is specified, approximately 40 nmoles of the first base are added to the DNA synthesizer. For an average 25-mer, at least 25% of this starting material will result in failure sequences; hence it is not possible to produce 40 nmoles of full-length product from a 40 nmole scale synthesis. The losses occur during synthesis, post-synthetic processing, transfer of material, and quality control. Final yield is the actual amount that we guarantee to deliver.

Please note that OD260 values are a measure of total nucleotides´ optical density. Hence, neither purity nor amount of ordered substance are transparently reflected. For simplification and exemplification reasons look at the following:

Therefore, a 1 OD guaranteed amount of delivered product can vary significantly, while metabion´s commitment to delivered yields in nmol does not allow for ambiguity in terms of what you expect and pay for.

DNA synthesis is a complicated process, which has improved significantly over the last years. Despite these improvements, all manufacturers have an inherent failure rate. We are constantly developing our processes and systems to minimize these losses; however, it is inevitable that we will occasionally have to re-synthesize some oligos. Please note that metabion performs strict quality controls on each and every oligo synthesized. If an oligo does not pass our quality tests, it will be resynthesized.

The expected average in-house turnover time for siRNA is 5-10 working days. Please note that we perform strict quality controls on each and every oligo. In case one or more oligos do not pass our quality control, they will need to be resynthesized. This may, of course, result in a delay.

In terms of "counting" working days, orders placed past 15:00 (Munich time) are considered to be next day's order.

Given the use of siRNA oligos in cells and in vivo, high purity is essential.

High pressure liquid chromatography (HPLC) purification is standard (no additional charge) for siRNAs. For applications in cells and in vivo, we recommend an additional SEC. This is a size exclusion chromatography purification that removes salts and other small molecules, whose quantity and/ or quality can be toxic for cells.

Unless requested, siRNA are synthesized with neither 3´nor 5´ phosphate. Both, the 5´and the 3´-end will carry a Hydroxyl-group. The 5´ phosphorylation of both strands is available as a modification at the additional cost of € 90,00.

There is a normal degree of variation in the appearance of the supplied dry pellets. Variation in appearance per se does not indicate a quality defect. In general, appearance of unmodified and dye-labeled siRNA pellets may vary from powdery to hyaloid. The color of unmodified siRNA pellets may range from transparent over off-white and yellowish to tan. The pellets of labeled siRNA are colored according to the dye attached.

We recommend to dissolve single stranded RNAs in 1x TE buffer (10 mM TrisCl, pH7.5, 0.1 mM EDTA; prepared under RNase-free conditions). This buffers the pH and chelates metal ions, which may contribute to RNA degradation. RNase-free water is also acceptable.

metabion's RNA oligonucleotides are delivered deprotected and purified – ready to use. They are RNAse free, but as RNA is highly susceptible to degradation by exogenous RNAses introduced during handling, it is essential that you conduct all handling steps under sterile, RNAse free conditions. Never handle RNA without wearing gloves. RNAse free reagents, barrier pipette tips and tubes should be used!

For more information, please read FAQ: How should siRNA oligonucleotides be stored?

RNA duplexes are significantly more resistant to nucleases than a single strand RNA oligonucleotide. However, maintaining sterile, RNAse free conditions is always recommended as a precaution. Dried pellets are stable at room temperature for 2-4 weeks, but should be placed at -20°C or -80°C for long term storage. Under these conditions, dry RNA is stable for at least one year. If you want to store your RNA in solution re-suspend the delivered pellet in an RNAse-free solution buffered at pH 7.4 - 7.6 and store at -20°C or less. We recommend that RNAs are re-suspended at a convenient stock concentration and stored in small aliquots to avoid multiple freeze thaw cycles.

Make sure your sequence is free of hairpins and self-complementary regions. These might affect both the synthesis and the hybridization of your oligo to the target.

More than six of the same consecutive bases (i.e. GGGGGGG) can be problematic and reduce final yields.

Modification PlacementWhenever possible, place modifications at the 5' end. Automated DNA synthesis occurs in the 3' to 5' direction. Each nucleotide addition is 98-99% efficient resulting in 1-2% of the oligonucleotide being truncated and capped at each position. Placing the modification at the 5' end ensures that only the full length oligo is modified. Furthermore, because most modifications are more hydrophobic than unmodified oligonucleotides, the full-length modified oligo binds more tightly to the reverse phase media during HPLC purification. This enhances the separation between the full-length, modified oligonucleotide sequences and the truncated, unmodified oligo sequences.

Purification MethodMetabion purifies each and every siRNA with HPLC. However, we strongly suggest to additionally purify your siRNA with a SEC, to minimize cytotoxicity.

Metabion is dedicated to reliably deliver high quality products. While every production step is performed in light of achieving best quality, the product is released only if it passes our final inspection. Mass Spectrometry has become the state-of-the-art technology for verifying the integrity of oligonucleotides, and metabion has been the first custom oligo house who introduced routine mass checks into its operations. Each and every oligo is characterized by either MALDI- or ESI-ToF and stringent release criteria are applied.

Mass Spectrometry allows for the most sensitive detection of low-level by-products/impurities such as

n-1/n-x oligos

Depurination

Incomplete Deprotection

Acrylonitrile adducts

High Salt Content Identification

Moreover, it is the fastest and most efficient way to identify potential product mix-ups.

We run two different types of Mass Spectrometry (MS) instruments in order to cope best with quality and quantity/throughput issues determined by the specifications of the respective oligo/analyte. While each instrument type precisely characterizes oligonucleotides in terms of composition through direct molecular weight measurement, their field of application is diligently adjusted to suitability considerations.

MALDI-ToF instruments typically have a higher throughput, while the limits of using this technique become manifest, if it comes to analyzing long oligonucleotides, or oligos carrying certain photo-labile modifications (e.g.common quenchers like BHQ®s, Dabcyl used in DLPs).

ESI-ToF is less efficient in terms of throughput but perfectly compensates for resolution issues with long oligos as well as for a potential detrimental laser impact on labile/photosensitive modifications – thus being a "natural" complement to MALDI-ToF analysis.

Comparison MALDI-ToF and ESI-ToF

Qualification Criteria

MALDI-ToF

ESI-ToF

< 60 nts

+

+

> 60 nts

-

++

Photosensitive Modified Oligos

-

+

Wobble Oligos

-

+

Throughput

++

+

n-1/n-x Detection

+

+

Incomplete Deprotection

+

+

Depurination

+

+

Mass Accuracy

++

++

Synthetic oligonucleotide purification is particularly challenging because of the small differences in size, charge and hydrophobicity between the full-length product and impurities, which often co-elute.

For improved analysis of complex samples like long and/or multiple labeled oligos, metabion offers liquid chromatography (LC) coupled with electrospray ionization mass spectrometry (ESI-MS). The mass spectrometer is connected to a high pressure liquid chromatography (HPLC) system, which allows premium analyte characterization via chromatographical separation, followed by respective molecular weight determination. With this system, the mass of oligonucleotides between 2 and 220 bases can be analysed with high accuracy, resolution and sensitivity. Our expert production team will take care of the method (MALDI or ESI ToF) that best applies to your sample.

Preparative High Pressure Liquid Chromatography (HPLC) deals with isolating the separated components of a sample, and can be done on small-, mid- and large scale operations. In other words, the objective of a preparative HPLC is isolating and purifying a product. Practically, the sample goes from the detector into a fraction collector or it is collected manually.

Analytical HPLC refers to the processes of separating and identifying the components of a sample. It is usually a small-scale process, whose objective is the qualitative and quantitative determination of a compound. The sample goes from the detector into waste.

metabion offers analytical HPLC as an additional (optional) quality control method, complementing our Mass-Check QC, which is performed by default on all our oligos.

For product/quality documentation please see FAQ: What kind of documentation do I get with my RNA oligos?

When you write your email, please make sure to address the following questions in the excel template:

Name of the siRNA?

Sequence of the siRNA in 5’-3’ orientation?

Yield range

Delivery form? (dry / in water/ buffer + concentration)

Modifications?

If you are a new customer, please additionally provide us with

Your shipping and billing address

Any other information like Purchase Order number, VAT number (VAT only for customers resident in the EU) etc

In case you opt to transmit orders via email using your own format(s), we need to alert you that above mentioned information are obligatory for processing your order. Due to extra efforts necessary for individual order format transfer into our system, order processing will take longer as compared to preferred web orders and pre-formatted emails.

The label on the siRNA tube shows basic information like oligo name, name of person who made the order, siRNA sequence including modifications, oligo ID, amount of RNA (OD260 and nmol), Tm, and molecular weight.

In addition, you will receive a technical data sheet containing more detailed information on the physical-chemical properties of the oligo, such as base composition, base count, purification grade, amount of RNA in nmol, Tm and molecular weight. Additionally, you will also receive a hard copy of the Mass-Check documentation/traces of each single strand of the duplex. The following terminology is used for differentiating between offered QC options including respective documentation coverage in our order forms and on supporting documents delivered with the products:

Mass CheckStandard quality control performed on each and every oligo. Either MALDI- or ESI-ToF, subject to the "nature of the oligo", and metabion internal procedures. This service is free of charge and the Mass Check traces will be provided.

While some manufacturers state that re-annealing is generally not necessary, we do recommend to re-anneal the dissolved duplex using a suitable protocol. For your convenience, we recommend to follow our guidelines prepared for download.

All oligonucleotides, whether single-stranded or double-stranded oligos, are provided as dried pellets and shipped at ambient temperature. While being stable at room temperature for 2-4 weeks, they should be placed at -20°C or at lower temperature upon receipt.